The long-term goal of the project is to identify targets for the effective prevention and treatment of biofilm-associated infection. We have selected an approach where an increase in our understanding of the bacterial physiology that underlies biofilm formation will lead to an improved ability to manipulate the bacteria. Using the enteric bacterium Escherichia coli as a model organism, a partial network of transcriptional regulation affecting biofilm-associated cell surface organelles was previously summarized. This proposal focuses on a portion of this network, including the flagellar and global regulator complex FlhD/FlhC and several two-component systems, such as EnvZ/OmpR and RcsCDB. Metabolic signaling molecules relating to acetate metabolism are included. The signaling pathways through these molecules are parallel in the upstream pathways and intersecting in the downstream ones. As a consequence, multiple environmental signals are integrated to one final outcome, the formation of biofilms. Most current studies investigate gene expression of the entire biofilm at one time point. This does not acknowledge that biofilms undergo a developmental process and that even the fully developed, mature biofilm consists of several distinct ecological niches. Our proposed research aims at determining the temporal and spatial distribution of gene expression during biofilm formation. We anticipate that this will be a major breakthrough in our understanding of the environmental control of biofilm formation. As a translational aspect, results from the proposed research will aid in the development of novel contact-active compounds that will be integrated into silicone based antimicrobial coatings and surfaces, a project that is done by collaborators at the Center for Nanoscale Science and Engineering at NDSU. Genes that are required for biofilm formation and expressed early in biofilm development will be of particular interest for this intriguing application. The basis for the Specific Aims is a data set of quantitative biofilm data that was obtained with many E. coli mutants under multiple combinations of environmental conditions. Using the environmental conditions that gave the largest differences in biofilm associated biomass between mutants in flhD, rcsB, ompR, and genes encoding enzymes of acetate metabolism, qualitative and quantitative assessment of biofilm formation will be performed with stationary biofilms and scanning electron microscopy, as well as continuous biofilms and confocal microscopy. This will constitute Specific Aim I. Combinations of mutations will be included to establish hierarchies among regulatory effects. In a second Specific Aim, promoters from some of the above genes will be fused to genes encoding fluorescence proteins and the temporal and spatial expression from the promoters will be investigated within the biofilms. This will be done in wild type bacteria and a selection of the mutants. The qualitative data will be related back to the environmental screening experiment by quantifying biofilms from the confocal microscopy images.